Methods for setting up, monitoring and performing analysis using capillary gel arrays

ABSTRACT

Set up methods, analysis methods and monitoring methods for capillary gel arrays are provided which rigorously determine the number of capillaries for which standards should be used and/or the capillaries in which standards should not be considered and/or for which performance is impaired. The invention includes methods for monitoring capillary gel array performance in which the capillary gel array performs, on a plurality of occasions, a size based separation on an unknown sample in one or more capillaries, a size based separation on a second standard in those one or more capillaries, and a size based separation on a standard in one or more other capillaries, comprising comparing the results of the sample to the results of the standards in which variation in a characteristic of the results from the capillaries provided with the standard are considered with time and variation of the characteristic outside a predetermined position to determine if one or more of the capillaries of the array are providing reduced performance. The invention provides rigour to the results obtained from capillary gel array analysis and prevents analysis being carried out on arrays which have developed problems or broken down.

This invention concerns improvements in and relating to analysis, inparticular, but not exclusively, in relation to capillary array gelelectrophoresis.

Capillary array gel electrophoresis is increasingly used in a variety ofDNA analysis techniques. It is becoming more commonly used in shorttandem repeat, STR, analysis due to its suitability for analysis oflarge numbers of samples in automated processes. STR analysis findsparticular use in medical and forensic applications due to its abilityto determine variations in the DNA of individuals. In forensic scienceit is used to establish profiles for individuals which can be comparedwith other profiles to establish a match or eliminate a match. Thecomparison may be with another sample or with established records of STRprofiles for individuals.

Whilst capillary array gel electrophoresis is finding increasing use theapplicant has established that there are a number of issues which couldcompromise the actual accuracy of the results or the validity of thoseresults particularly in legal proceedings. Any question mark over thevalidity of a DNA profile or its comparison could be exploited in legalproceedings to question its evidential value. The present invention hasamongst its aims the provision of results which are not subject to theseissues.

The performance of capillary array gels with time can also vary and evenbreakdown, leading to wasted tests being performed before such problemsare realised. The present invention has amongst its aims the monitoringof performance of arrays with time and the provision of warnings inadvance of breakdown.

According to a first aspect of the invention we provide a method forsetting up a capillary gel array for analysis use, in analysis use thecapillary gel array performing a size based separation on an unknownsample in one or more capillaries and a size based separation on astandard in a number of other capillaries, the results of the one ormore unknown samples being compared with the results from the one ormore standards to obtain the analysis results,

which set up method determines the number of capillaries in which thesize based separation on a standard should be performed in analysis use,the set up method including, for one or more components of the standard:

determining a target standard deviation of the mean size for thecomponent;

determining, by multiple size based set up separations, a relationshipbetween the experimental standard deviation of the mean size for thecomponent and the number of capillaries used for size based separationsof the standard; and

with the experimental standard deviation equating to the target standarddeviation indicating, by means of the relationship, a boundary number ofcapillaries, the number of capillaries being used for size basedseparation of the standard in analysis use being a whole number and atleast as great as the boundary number.

Preferably the set up method is used before any analysis use of thearray. Preferably a set up method may be used for each array used on aninstrument. The set up may be employed when the standard being used inthe analysis method is changed.

Preferably the determination is of the minimum number of capillarieswhich should be used for size separation of standards, ideally whilstbeing statistically reliable to a given degree of confidence. Preferablythe minimum number is used in the analysis use, ideally with all othercapillaries being provided with unknowns.

The set up method may be performed on only one component of thestandard. The components may be include a component of the heaviestweight loci present in the standard. The component may be or include theheaviest weight component of the standard, ideally the heaviest weightcomponent of the heaviest weight loci present in the standard. The onecomponent may be or include a component of the most AT rich loci presentin the standard. The one component may be or may include the most ATrich component of the standard. The most AT rich may be the componentwith the most AT bases in total and/or the component with the highestproportion of AT bases in its sequence.

Preferably the one or more components includes the component with thehighest standard deviation of mean size in experimental measurements ofits size, particularly capillary gel electrophoresis measurements.

The target standard deviation may be based on one or more factors. Thefactors may include the tolerable discrepancy between the actual meanand experimentally determined mean size for that component and/or thedegree of confidence required and/or the distribution type allocated tothe allele size experimentally determined. The tolerable discrepancy ispreferably +/−0.25 bases. The degree of confidence is preferably atleast 95%, more preferably at least 98% and ideally at least 99%. Thedistribution type is preferably a normal distribution, but others may beprovided.

The target standard deviation is preferably less than 0.1, morepreferably less than 0.09 and ideally less than 0.083, particularly fora 96 capillary array and/or a normal distribution and/or a degree ofconfidence of 99% and/or a tolerance of +/−0.25 bases.

The target standard deviation and/or tolerable discrepancy and/ordistribution type and/or degree of confidence may be different fordifferent standards and/or numbers of capillaries in the array.

Preferably a plurality of set up size based separations are conductedfor a component with a given number of capillaries provided withstandard. Preferably the plurality is at least 25 times, more preferablyat least 100 times, still more preferably at least 250 times and ideallyat least 1000 times for each number of capillaries provided with thestandard. Preferably a plurality of set up based size separations areprovided for each of between 1 and 5, more preferably each of between 1and 10 and ideally each of between 1 and 20 capillaries provided withthe standard.

Preferably the relationship is the variation of standard deviation ofthe mean size of the component, experimental, with the number ofcapillaries provided with the standard.

Preferably the an experimental standard deviation value equivalent tothe target standard deviation value is taken and applied to therelationship. Preferably the application of the experimental standarddeviation relates to a number of capillaries provided with standard.Where the number is a whole number preferably that number of capillariesare provided with standard in the analysis use. Where the number is anon-whole number, preferably that number is rounded up to give thenumber of capillaries provided with the standard in analysis use.

The number is preferably a number between 2 and 6, more preferablybetween 3 and 5, still more preferably 4 or 5 ideally 4, particularlyfor the Applied Biosystems AMPFISTR SGM PLUS system DNA profilingtechnique and/or HUMFIBRA locus and/or a 96 capillary array. Othernumbers may arise for other multiplex systems for analysing STRs.

According to a second aspect of the invention we provide a method ofanalysis DNA in a sample using a capillary gel array, the use thecapillary gel array performing a size based separation on an unknownsample in one or more capillaries and a size based separation on astandard in a number of other capillaries, the results of the one ormore unknown samples being compared with the results from the one ormore standards to obtain the analysis results,

the set up method for which includes determining the number ofcapillaries in which the size based separation on a standard should beperformed in analysis use, the set up method including, for one or morecomponents of the standard:

determining a target standard deviation of the mean size for thecomponent;

determining, by multiple size based set up separations, a relationshipbetween the experimental standard deviation of the mean size for thecomponent and the number of capillaries used for size based separationsof the standard; and

with the experimental standard deviation equating to the target standarddeviation indicating, by means of the relationship, a boundary number ofcapillaries, the number of capillaries being used for size basedseparation of the standard in analysis use being a whole number and atleast as great as the boundary number.

The second aspect of the invention may include any of the features,options or possibilities set out elsewhere in this document, withparticular reference to those set out in the first aspect of theinvention and related statements.

According to a third aspect of the invention we provide a method forsetting up a capillary gel array for analysis use, in analysis use thecapillary gel array performing a size based separation on an unknownsample in one or more capillaries and a size based separation on astandard in one or more other capillaries, the results of the one ormore unknown samples being compared with the results from the one ormore standards,

in which set up method a group of capillaries at one or both ends of thearray are excluded from use as capillaries for the standard insubsequent use of the array.

Preferably a group of capillaries from both ends of the array areexcluded. The group of capillaries may be sequential in terms of thecapillaries which are excluded. Preferably at least the first capillaryor the last capillary of the array is excluded and ideally both.

One or both groups may include at least two or more preferably at leastthree capillaries. One or both groups may include between 2 and 4capillaries in them and more preferably include three capillaries.

Preferably the excluded capillaries are used in analysis use for unknownsamples.

The excluded capillaries may be additional to those excluded accordingto the method of the fifth aspect of the invention or may include one ormore common capillaries.

Preferably a set up method of this type is performed on each arrayand/or each capillary array instrument before analysis use.

According to a fourth aspect of the invention we provide a method ofanalysing DNA containing samples using capillary gel arrays in which thecapillary gel array performs a size based separation on an unknownsample in one or more capillaries and a size based separation on astandard in one or more other capillaries, the results of the one ormore unknown samples being compared with the results from the one ormore standards to provide the analysis results,

in which set up method a group of capillaries at one or both ends of thearray are excluded from use as capillaries for the standard insubsequent use of the array.

The fourth aspect of the invention may include any of the features,options or possibilities set out elsewhere in this document, withparticular reference to those set out in the third aspect of theinvention and related statements.

According to a fifth aspect of the invention we provide a method forsetting up a capillary gel array for analysis use, in analysis use thecapillary gel array performing a size based separation on an unknownsample in one or more capillaries and a size based separation on astandard in one or more other capillaries, the results of the one ormore unknown samples being compared with the results from the one ormore standards,

in which a size based separation is performed in each of a plurality ofthe capillaries on a known material to determine a speed of migrationrelated characteristic of the known material in those plurality ofcapillaries, those capillaries from amongst the plurality of capillarieswhich have a characteristic value outside a predetermined range beingexcluded from use as capillaries for the standard in subsequent use ofthe array.

Preferably the size based analysis of the set up method is performed onall the capillaries of the array. Preferably the same known material isused in each capillary. Preferably the characteristic value isdetermined by the distance the material has migrated in each of thecapillaries in the same time period. The characteristic may be a speedof migration and/or a distance of migration and/or a size associatedwith that degree of migration. The size may be expressed in terms ofbases.

The predetermined range may be defined by an upper limit. Thepredetermined range may be defined by a lower limit. The limits may bedefined in absolute terms. The absolute terms may be exclude the highestfew and/or lowest few characteristic values. The few may be one, two oreven three in each case. The limits may be defined relative to astandard value, for instance the mean or median value. The relativedefinition may be provided as a +/−%. A range of+/−0.1% may be appliedor a range of +/−0.075% may be applied, as the limit of the range,particularly when expressing the characteristic as relative sizes interms of bases. The relative definition may be provided as a standarddeviation relative to a standard value.

Preferably the excluded capillaries are used in analysis use for unknownsamples.

The excluded capillaries may be additional to those excluded accordingto the method of the second aspect of the invention or may include oneor more common capillaries.

Preferably a set up method of this type is performed on each array priorto analysis use.

If a characteristic is outside a further predetermined range, then thecapillary may be excluded from use as capillaries for an unknown samplein subsequent use of the array. The further predetermined range may be arange of +/−0.5%, may more preferably be a range of +/−0.25% and may bea range of +/−0.15%, as the limit of the range, particularly whenexpressing the characteristic of relative sizes in terms in bases. Thefurther predetermined range may be +/−5% of the distance of migrationand/or speed of migration compared to the mean.

According to a sixth aspect of the invention we provide a method ofanalysing DNA containing samples using capillary gel arrays in which thecapillary gel array performs a size based separation on an unknownsample in one or more capillaries and a size based separation on astandard in one or more other capillaries, the results of the one ormore unknown samples being compared with the results from the one ormore standards to provide the analysis results,

and in which a size based separation is performed in each of a pluralityof the capillaries on a known material to determine a speed of migrationrelated characteristic of the known material in those plurality ofcapillaries, those capillaries from amongst the plurality of capillarieswhich have a characteristic value outside a predetermined range beingexcluded from use as capillaries for the standard in subsequent use ofthe array.

The sixth aspect of the invention may include any of the features,options or possibilities set out elsewhere in this document, withparticular reference to those set out in the fifth aspect of theinvention and related statements.

According to a seventh aspect of the invention we provide a method ofmonitoring capillary gel array performance in which the capillary gelarray performs, on a plurality of occasions, a size based separation onan unknown sample in one or more capillaries and a size based separationon a standard in one or more other capillaries, the results of the oneor more unknown samples being compared with the results from the one ormore standards,

in which variation in a characteristic of the standard results isconsidered with time and variation of that characteristic outside apredetermined position provides information on the array.

The performance may be the reliability of the results. The performancemay be the migration speed of a standard in a capillary with time.

The plurality of occasions may be greater than 10 occasions, morepreferably greater than 50 occasions, still more preferably greater than100 occasions and even greater than 1000 occasions.

The characteristic may be the distance of migration of one or morecomponents of the standard. The characteristic may be the speed ofmigration of one or more components of the standard. Preferably thecharacteristic is a standard deviation. The standard deviation may be ofthe speed of migration and/or distance of migration and/or morepreferably the size of component, for one or more components of thestandard.

The variation may be a change in the characteristic, particularly achange which causes the characteristic to cross a threshold. Thethreshold may be of an absolute value, for instance a preset speed, apreset distance or a preset standard deviation. The threshold may be arelative value.

The variation may be a change in the rate of variation of thecharacteristic, particularly a rate of change which exceeds a threshold.The threshold may be absolute or relative.

The variation may be determined after an occasion of use. The variationmay be determined after each occasion of use or more preferablyperiodically. The variation may be determined from the results arisingfrom the use. In particular the variation may be determined from theresults for the standard obtained from one or more of the capillaries ofthe array, most preferably those standards being used in the comparisonwith the unknown samples in the occasion. The variation may bedetermined from a separate set of results to those arising from theoccasions of use. In particular the separate set of results maybeobtained by running the array with the standards in one or morecapillaries of the array, ideally specifically for the purpose ofmonitoring. Ideally in this case the standard is run in all thecapillaries of the array.

The information on the array may be an indication that one or morecapillaries are providing reduced performance. The information may bethat one or more capillaries are not functioning within requiredparameters, for instance the capillary is too fast or too slow. Theinformation may be a warning that performance of one or more capillariesis approaching the limit of required parameters for proper operation,preferably the warning is provided before the parameters are crossed andideally in time to allow array replacement before the parameter arecrossed. Preferably the information results in the change of the array.

According to an eighth aspect of the invention we provide a method ofanalysing DNA containing samples using capillary gel arrays in which thecapillary gel array performs, on a plurality of occasions, a size basedseparation on an unknown sample in one or more capillaries and a sizebased separation on a standard in one or more other capillaries, theresults of the one or more unknown samples being compared with theresults from the one or more standards to provide the analysis results,and

in which variation in a characteristic of the standard results isconsidered with time and variation of that characteristic outside apredetermined position provides information on the array.

The eighth aspect of the invention may include any of the features,options or possibilities set out elsewhere in this document, withparticular reference to those set out in the seventh aspect of theinvention and related statements.

According to a ninth aspect of the invention we provide a method ofanalysis DNA in a sample using a capillary gel array, in use thecapillary gel array performing a size based separation on an unknownsample in one or more capillaries and a size based separation on astandard in a number of other capillaries, the results of the one ormore unknown samples being compared with the results from the one ormore standards to obtain the analysis results, in which between 3 and 8of the capillaries are provided with the standard.

Preferably between 3 and 6, more preferably 4 or 5 and ideally 4 of thecapillaries are provided with the standard.

The standard may include the locus HUMFIBRA.

The capillary array may include 96 capillaries.

The ninth aspect of the invention may include any of the features,options or possibilities set out elsewhere in this document.

The first and/or second and/or third and/or fourth and/or fifth and/orsixth and/or seventh and/or eighth and/or ninth aspects of the inventionmay include any of the features, options or possibilities set outelsewhere in this document including the following.

The capillary gel array preferably provides at least 10 capillaries,more preferably at least 25, still more preferably at least 36capillaries and ideally in excess of 50, for instance at least 86capillaries.

The array preferably includes a series of capillaries provided with gel.The capillaries are preferably parallel to one another. The capillariesmay be arranged in a linear array.

The analysis use preferably involves analysing one or more unknownsamples. Preferably a different sample is analysed in each capillary notprovided with the standard. The unknown sample may be provided to thecapillary along with a second standard in one or more, and preferablyall cases. The second standard may be formed of a series of concatamersof known size, ideally dye labelled, for instance with a red dye.

The size based separation is preferably achieved by electrophoresis.

The standard is preferably an allelic ladder. The standard is preferablyformed of a series allelic fragments. Preferably the standard includes anumber of fragments relating to at least some of the variations possibleat each of the loci under consideration. The loci may be as set outbelow.

The comparison of the unknown sample results and the standard resultsmay involve deciding that a component of the unknown sample isequivalent in identity to a component of the standard where the unknowncomponent position is within a positional range relative to thecomponent of the standard. The positional range may be +/−0.5 bases.Preferably the comparison between unknown sample and standard idperformed for each component.

The analysis results may be used as, or as part of, a DNA profile of thesample. The profile may thus be related to an item and/or locationand/or individual. The profile may be used to link and/or exclude thesample with another sample or profile arising therefrom.

Various embodiments of the invention will now be described by way ofexample only and with reference to the accompanying drawings, in which:

FIG. 1 shows a comparison of allelic ladder marker with questionedsample;

FIG. 2 is a plot of standard deviation across a 96× capillary arrayagainst size of allelic ladder markers (the loci are abbreviated asfollows: D21S11 and HUMFIBRA, HUMTH01, D19S433, D18S51, D21S11, D8S1179,Amelogenin, D2S1338, D16S539, HUMVWFA31, D3S1358);

FIG. 3 illustrates a typical frequency distribution for allele sizemeasured for HUMFIBRA 47.2 across 96 capillaries;

FIG. 4 presents Table 1 which is a comparison of standard deviations forHUMFIBRA alleles across five different arrays;

FIG. 5 a illustrates that the error distribution of an allele can occupya maximum one dimensional space or bin of 1 base, where A is the actualmean and B is the estimate of the mean, but that if the estimate of A isskewed then the 1-base bins may overlap and therefore there is anincreased chance of the band falling into the wrong (adjacent) bin;

FIG. 5 b illustrates that if the error distribution is a range of just0.5 base then any estimate B will be within range of a 1 base bindefined by A;

FIG. 6 illustrates the standard deviation of the mean and medianestimates relative to the number of allelic ladders run across a 96×capillary array for ladder markers HUMFIBRA in respect of alleles 26 and47.2; and

FIG. 7 illustrates the fragment size determined for fragments HUMFIBRAallele 47.2 relative to the capillary no. recorded from left to rightfacing an instrument and hence the variation in migration speed as thesamples are the same in each capillary.

BACKGROUND

In forensic science, short tandem repeat analysis, is frequently used toprofile an individual and/or sample from a location or an item with aview to marching that profile to another or to determining a non-matchso as to eliminate a link. Applied Biosystems AMPFISTR SGM PLUS systemDNA profiling technique, for instance, is a single multiplex reactionused to PCR amplify ten STR's and amelogenin (for gender determination)using fluorescent labeled primers. The discriminating power in forensicapplications using such multiplexes is such that the chances of twounrelated people having the same profile is approximately 10⁻¹³.

The analysis involves collection and preparation of the sample, PCRamplification using the multiplex and separation of the productsaccording to their size using one of a number of techniques. Separationusing capillary gel array electrophoresis, CE, is becoming increasinglypopular due to its ability to analysis a large number of samples in ashort time period, the removal of the need to manually produce the geland in a manner suited to automation.

The multiplexes used are designed so that the alleles of different lociwhich are labelled with the same colour dye do not overlap with oneanother so that each STR can be determined by its position and colour.The size fragments in the above example range from 100 to 360 bases andfour different coloured dyes are used.

As well as the dyes associated with the multiplex products, a redlabelled standard size marker having bases ranging from 50 to 400 isprovided. This is formed of concatamers with constant ACTG proportions,and is run in the same lanes/capillaries as the samples beingconsidered.

Comparison of the position of the sample products, Q samples, with thestandard size marker forms the first part of the sizing process. Thesecond part involves comparison with an allelic ladder formed of dyelabelled known size and sequence fragments. The limited number ofsuitable dyes means that the allelic ladder has to be run in a separatelane, or in the case of CE, separate capillary, to a Q sample.

Theory of Size Determination

Both the allelic ladder markers and unknown or questioned (Q) allelesare sized relative to an internal set of DNA markers such as HD-400 ROXstandards. The size of an allele (in bases) is always estimated relativeto sequenced standards that comprise control allelic ladder markers.

Because all size measurements are made relative to the allelic ladder,determination of the absolute size is not important since comparisonsare made directly against a control of the same size and sequence—henceit is only the distance of separation between control allelic laddermarker and the Q allele that is important. This is an importantconsideration because different internal size standards from differentmanufacturers will give different absolute results. Consequently, it isonly necessary to standardise allelic ladders.

Provided that the size of an allele is no more than 0.5 bases from themeasured allelic ladder control standard then a designation is safe tomake. In any electrophoretic system distortion of the run may also occurand this can result in band shift which occasionally pushes a band intothe next ‘bin’. To capture these events, Gill et al [Int. J. Legal Med.1996, 109, 14-22] also introduced a series of rules based on measurementof band shift relative to the allelic ladder marker which are explainedwith reference to FIG. 1. In particular:

-   -   a) The sizes in bases of questioned allele Q₁ and allelic ladder        allele (x) are measured relative to the internal standard,        usually by using the Elder and Southern [Anal. Biochen:. 1983,        128, 227-231] local method of measurement. This method        calculates the size of the Q allele relative to the two adjacent        internal size standards either side of it. Measurements are        repeated with Q₂ and allelic ladder allele)). Delta (d) values        are always conditioned on the allele where both the allelic        ladder marker and Q alleles are coincident within the same 1        base bin. The difference in sizes of the questioned and allelic        ladder markers are defined as:        d ₁ |x=fQ ₁ −fx and d ₂ |y=fQ ₂ −fy where f=size in bases.        Hence d₁|x and d₂|y must always be less than ±0.5 bases in order        to be designated.    -   b) The band shift association rule states that if one band of a        heterozygote is shifted, then the other allele will also be        shifted in the same direction and to the same extent. If d₁ and        d₂ are the respective band shifts for heterozygous alleles Q₁        and Q₂ relative to allelic ladder markers x and y respectively        then the allele designation is made only if        d ₁ |x−d ₂ |y<0.5    -   c) If a heterozygote comprising 2 rare alleles is observed, then        this observation must be confirmed by re-analysis (band shifts        will usually shunt the alleles into adjacent bins corresponding        to alleles (x±1 and y±1) that are usually occupied by alleles        that are rare. These rules can be programmed into expert        systems.        Problems

In flat bed gel electrophoresis a single allelic ladder lane is used fora gel slab which may have a large number of Q sample lanes run on it. InCE the convention is also to run one capillary of the array with anallelic ladder and the other capillaries with Q samples to maximise thenumber of Q samples which can be processed in a given time. Theapplicant has determined that this approach is not appropriate for theresults to be of the utmost validity.

Because it is not possible to include internal allelic ladder markerswithin each capillary (because of insufficient dyes and cost) thecomparisons of the allelic ladder with Q samples are always made betweendifferent gels in CE. In effect each capillary is a different gel. As aresult the applicant has realised that the impact of the present systemof using only one allelic ladder in a set of 96 capillaries can lead tosubstantial question marks over the designations applied to individualresults from the 95 capillaries containing samples under analysis. Amethodology has therefore been developed to establish the number ofallelic ladder containing capillaries which should be run to ensure thatthe designations are correct in an absolute sense and also to giveconfidence that the designations offer the necessary level ofstatistical confidence when scrutinised as forensic evidence, in a courtof law for instance. Even with relatively small numbers of capillariesin the array, such as 16, the necessary number of allelic laddercontaining capillaries needs to be verified.

Deviations in performance between one capillary and another arise for avariety of reasons.

Silica capillaries possess an excess of negative charge on theirsurfaces. Cations from aqueous solution build up at these surfaces, andwhen a charge is applied the cations, in the solution, are attractedtoward the cathode. This results in a bulk flow toward the cathode,which is against the migration of the DNA, causing disruption toseparation and consequent reduction of resolution of DNA fragments. Thecommonest method to reduce EOF is to coat the capillary inner-surface inorder to modify or mask the charge on the silica surface, but the can bevariations in the modification which results. Variations in the level ofmodification also apply to more sophisticated systems such as AB's useof a “dynamic coating polymer” POP-6 (Performance Optimised Polymer)Because the separation of the molecules, whether it be due to thetransient entanglement mechanism and/or by non-tangling collisionsbetween the DNA and polymer, is proportional to the size of themolecule, and because the mobility of DNA is also sequence dependent(notably, AT—rich sequences show anomalous migration rates relative tointernal size standards) it is recommenced that the evaluation of thenumber of allelic ladders required is conducted in relation to the locishowing the greatest standard deviation against the size standards intest. The philosophy is preferably to carry out evaluation on worst casescenarios. Using this principal the methods and logic described shouldbe applicable to any capillary array system whatever the number ofcapillaries or loci being considered apply.

Experimental Demonstration of Variation

The methodology followed in analysing the samples and obtaining standarddeviation data is set out in Appendix 1 below.

Allelic ladders were run across an entire 96× array and the standarddeviations (SD's) of each allele were compared, FIG. 2. Interestingly,different loci have different characteristics. The standard deviation isboth locus dependent and dependent upon the molecular weight ofindividual alleles; the standard deviation increases with the molecularweight. The data form three distinct clusters a) low SD: D2S1338,D16S359, D21S11, HUMVWFA31/A, HUMTH01, D19S433 b) High SD:HUMFIBRA(FGA), D8S1179, D3S1358; c) intermediate SD: D18S51. The highmolecular weight alleles of the HUMFIBRA locus show the greatest SD,followed by D18S51. All other loci have much lower SD's.

The repeating sequences of the high SD loci HUMFIBRA, D8 and D3 areapproximately 75% AT-rich. D18S51, which also has an elevated standarddeviation, is 75% AT-rich. The only locus that does not fit this patternis D16, which is part of the low SD cluster.

Worst case scenarios are defined as alleles that are most likely to falloutside their 1-base bin (as discussed in the theory of size analysisabove) and these can be evaluated using high molecular weight HUMFIBRAalleles because they have the highest SD's—the range for a highmolecular weight HUMFIBRA allele was approximately 1.25 bases, see FIG.3, with a maximum SD of 0.16, see FIG. 4 which provides Table 1.

Determination of Number of Allelic Ladders Needed

Referring to the sizing theory, a ‘bin’ of ±0.5 bases is constructedaround the observed position of an allelic ladder allele. Supposing thatthe range of measurement error is also one base then provided that Qalleles fall within this bin then they are correctly designated.However, this will be entirely dependent upon the measurements of theestimate of the mean (B) being coincident with the actual mean (A).

However, if the bin has been constructed from an observation (B) that isin the tail of the error distribution, see FIG. 5 a, then the one basebin construct will overlap into an adjacent bin, (portion C) and it ispossible therefore that Q alleles that actually should be designated inthe next bin could appear to fall in the bin constructed aroundobservation (B) and could be mis-designated as a result.

However, if the maximum measurement error range is set at just ±0.25bases, centred on the estimate of the mean (B), see FIG. 5 b, then evenif the estimate (B) is taken from the tail of the measurement errordistribution of (A) the Q alleles will always fall within the correctbin because the ±0.25 bases bin around (B) will always be within the±0.5 bases bin around (A). This means that effectively, the range shouldbe no greater than 0.5 bases in total if mis-designation is to beavoided completely.

Against this position the possibility of error is therefore minimised byproviding a best estimate of the mean (B)and this is dependent upon thenumber of allelic ladders that are run across the 96× array in order tomake such estimates. The present position in the prior art is that asingle run of the allelic ladder is used to determine (B). Of course themore capillaries which are used for allelic ladders the better theestimate of (B), but the more allelic ladders that are used, the lowerthe number of samples that remain for analysis.

As a consequence of this position it is necessary to establish theminimum number of capillaries which need to be used for allelic laddersto achieve the necessary degree of confidence. To achieve thissimulation was used to determine the relationship between the standarddeviation and the number of allelic ladders run across the array. Foreach simulation a constant in allelic ladders were chosen at random,with replacement, from the array of 96 capillaries (where n=1 to 20) andthe experiment was repeated 1000 times so that a large sample ofestimates of the mean and median were generated from a single arraydata-set. The whole simulation was repeated by changing the value of it.Standard deviations were calculated from the 1000 estimates of the meanand median for each value on n.

The worst case scenario was evaluated specifically with the highmolecular weight HUMFIBRA allele 47.2 (MW=328). Ideally, as identifiedabove, the range should be less than 0.5 bases and this corresponds to acritical SD<0.083 (the critical SD is 0.5/6—under the assumption thatwith a normal distribution 99% of observations should be covered in abin of width 6×SD). Standard deviations of means and medians from the1000 simulations are shown in FIG. 4. As can be seen from FIG. 4, thecritical standard deviation corresponded to that calculated from 4allelic ladders for this high molecular weight allele. Standarddeviations of the mean were marginally lower than standard deviations ofthe median estimate. The simulation experiments were repeated withallele HUMFIBRA 26 (molecular weight 253). The critical SD was achievedwith just 2 allelic ladders with this lower molecular weight allelemarker, see FIG. 6.

Similar determinations of the minimum number of alleles needed can bemade for different loci, for different particular alleles or fordifferent levels of certainty. The determination can also be made fordifferent assumed distributions of the mean, other than normal. Thedetermination can be made in a similar way also for capillary arrayswith different numbers of capillaries.

Array and Capillary Specific Variations

The investigations behind this work also established that othervariations in the capillary array could make a significant impact on theaccuracy and/or robustness of the determinations made from the results.

When the differences in five consecutive runs were compared for HUMFIBRA47.2 it was noted that there were a number of consistent behaviours interms of speed of migration. As far as the 12 capillaries wherefragments migrated the fastest in these tests were concerned it wasnotable that capillary no. 1 (the left hand capillary when viewed fromthe front) always migrated the fastest. Capillaries 2, 3 and 4 wherealso included in the list, reinforcing the trend that the capillariesthat migrated the fastest were those that are low number. However, it isnoticeable that fragments in capillaries 55, 62, 67 also migrated fasterthan expected from the trend and furthermore that this was areproducible effect between different runs on the same array. Thiseffect was not reproducible when a new capillary array was implemented,however, and so each array requires separate evaluation for the fasteror slower mid array capillaries. The higher number capillaries, however,where consistently among the slower.

The implication of this position is that the systematic variation inspeed across the array points towards the slower and faster capillariesbeing avoided for use as the allelic ladder bearing capillaries. Thismeans that care needs to be taken in the choice of capillaries used torun the allelic ladders. If capillaries are all chosen from eitherextreme—capillaries 1-10 or 86-96 then the calculated means will tend tobe continually over or underestimated. Ideally, ladders should be chosenfrom the mid positions on the array, subject to the further observationsmade below, to reduce further the chance of a mis-designating alleles.Assessment of each CE machine is advisable to establish variations ofthis type.

Referring to FIG. 7 it is clear that within a particular array thatdifferent capillaries will give different individual performance, overand above any effect of the left to right/fast to slow variation.Capillaries which give fast or slow speeds from the middle part of thearray should also be avoided, therefore, for the allele laddercapillaries. As these performance variations effect differentcapillaries n different arrays it is recommenced that the set upprocedure for an array include an evaluation of the speed of theindividual capillaries and that the fastest few and slowest few (orthose exhibiting performance above or below a threshold) being excludefor use as the allelic ladder capillaries.

To assess each machine separately and each new array separately it isrecommended that each machine is characterised by running 96 allelicladders across the array in order to characterise the separatecapillaries and to ensure that those chosen to run allelic ladders givea mean result that is reasonably close to the ‘true’ mean.

Variation with Extent of Use

As well as inherent variation in speed for a CE machine, a CE array andindividual capillaries within an array, variation with time occurs.Indeed a point is reached with Ce arrays at which one or more of thecapillaries is no longer functioning and the results produced are of nouse and the tests need to be repeated. Unfortunately as a significanttime period often elapses between a test run being performed and theresults being analysed (at which time the breakdown of the array isnoticed) very substantial numbers of further tests will have been donein the meantime and these will need repeating (with consequent time andcost implications).

To avoid this the applicant suggests one or both of the followingmonitoring routines for CE arrays.

Firstly, by recording allelic ladder standard deviations between runsfor arrays as a whole or more preferably individual capillaries thevariation in the standard deviation with time can be established. SDlevel above a threshold can be used to warn of array breakdown andpromote shifting to a new array. Secondly, a similar aim can be achievedby carrying out a full analysis by running allelic ladders across theentire array at regular intervals. The performance of an array can bemonitored by direct reference to the standard deviation—it would beexpected that the standard deviation would increase if the array startsto break down, acting as an early warning indicator of a problem.

The methodology set out in the present invention: in determining theeffective number of allelic ladders which should be used; in determiningwhich capillaries to avoid for the allelic ladders due to machine orarray variations; in determining which capillaries to avoid for allelicladders due to capillary specific variations; and in providingforewarning of array breakdown offers technology which provides moreeffective CE analysis through more rigorous results and improvedprocessing efficiency.

Appendix 1

For sample preparation the DNA was extracted from buccal scrapes usingQIAAMP spin columns (Qiagen)as described by Greensppon et al. [J.Forensic Sci. 1998, 43, 10-24-1030] and was PCR amplified using a STRmultiplex from Applied Biosystems (AB) AMPFISTR AG Plus DNA profilingtechnique, as described by Cotton et al [Forensic Sci. Int. 2000, 112,151-161] for use on the AB 377 flat-bed gel automated sequencer. Aconcatamer internal size standard AB HD400 Rox was included with everysample-this included the following fragment sizes: 50, 60, 90, 100, 120,150, 160, 180, 190, 200, 220, 240, 260, 280, 290, 300, 320, 340, 360,380, 400 base pairs. In addition the SGM plus allelic ladder sizestandard was incorporated in to 8 capillaries per array run. The allelicladder cocktail comprises alleles from the following loci using filterF.

Dyes are 5-FAM (blue); JOE (green); NED (yellow): D3S1358 (blue);HUMVWFA31/A (blue); D16S359 (blue); D2S1338 (blue); Amelogenin (green);D8S1179 (green); D21S11 (green); D18S51 (green); D19S433 (yellow);HUMTH01 (yellow); HUMFIBRA(FGA) (yellow).

The STR loci utilised are tetrameric repeat sequences. Alleles in theladders encompass the entire range of common alleles and are spaced at aminimum of 4 bases apart and coincide with the common alleles. InHUMFIBRA(FGA); D19S433 and D21S11 2 base variants are common; hence 2base variants are included with the ladders for these loci.

The loading buffer (Applied Biosystems) HIDI Formamide was used.

Size Standard was diluted in HIDI Formamide at 1 in 40 ratio. 1.5 ml PCRProduct+13.5 ml HIDI Formamide/HD400 Size Standard. 1.5 ml Allelicladder+10 ml HIDI Formamide/HD400 Size Standard.

Electrophoresis was conducted on the ABI Prism 3700 CE platform usingstandard run parameters. Labelling and sizing of DNA fragments and theirallelic designation was carried out with Genotyper 1.1.1 software.

The separation matrix used was POP-6 (Performance OptimisedPolymer-6[7]) using 1×TBE running buffer (supplied by AB). The sampleswere injected from AB Gene Microtitre plates or ABI ThermofastMicrotitre plates. A sample transfer volume of 2.5 ml usingelectrokinetic injection parameters of 10 kV for 11 secs, a run voltageof 7.5 kV, run temperature of 50° C.; cuvette temperature between 45° C.to 50° C. (note this temperature must be optimised for each separate3700 machine otherwise sensitivity is compromised); the cuvette polymerflow rate is 12000 counts.

1. A method of monitoring capillary gel array performance between two ormore uses of the capillary gel array on an instrument, the method ofmonitoring comprising: performing a use of the capillary gel array andperforming a subsequent use of the capillary gel array, wherein a use ofthe capillary gel array is defined as including the capillary gel arrayperforming a size based separation on an unknown sample in one or morecapillaries, a size based separation on a second standard in those oneor more capillaries and a size based separation on a first standard inone or more other different capillaries, results of the one or moreunknown samples being compared with results from the one or morecapillaries provided with the first standard and results of the one ormore unknown samples being compared with results from the one or morecapillaries provided with the second standard; determining variation ina characteristic of the results from the capillaries provided with thefirst standard between two or more uses of the capillary gel array;using variation of that characteristic outside a predetermined positionto provide information on the capillary gel array, the information beingthat one or more of the capillaries of the array are providing reducedperformance; determining the timing of the provision of a replacementcapillary gel array using the information, the capillary gel array beingreplaced by the replacement capillary gel array; and replacing thecapillary gel array on which the use and subsequent use were performedwith the replacement capillary gel array according to the timingdetermined.
 2. A method according to claim 1 in which the standard has amigration speed or a migration distance and the performance monitored isthe migration speed or migration distance of the standard in a capillarywith time.
 3. A method according to claim 1 in which the standard has amigration speed or a migration distance and the characteristic is thedistance or speed of migration of one or more components of thestandard.
 4. A method according to claim 1 in which the standard has amigration speed or a migration distance or a size of component and thespeed of migration and/or distance of migration and/or the size ofcomponent for one or more components of the standard is thecharacteristic and the characteristic is expressed as a standarddeviation.
 5. A method according to claim 1 in which variation is achange in the characteristic which causes the characteristic to cross athreshold.
 6. A method according to claim 5 in which the threshold is anabsolute value and is a preset speed or a preset distance or a presetstandard deviation.
 7. A method according to claim 1 in which thevariation is a change in the rate of variation of the characteristic. 8.A method according to claim 1 in which the variation in thecharacteristic is determined after an occasion of use.
 9. A methodaccording to claim 1 in which the variation is determined from theresults for the standard obtained from one or more of the capillaries ofthe array.
 10. A method according to claim 1 in which the information onthe array is an indication that one or more capillaries are notfunctioning within required parameters.
 11. A method according to claim1 in which the information on an array is a warning that performance ofone or more capillaries is approaching the limit of required parametersfor proper operation.
 12. A method according to claim 1 in which thecapillary gel array is also used in a method of analysing DNA containingsamples, the results of the one or more unknown samples being comparedwith the results from the one or more capillaries provided with thestandard and being compared with the second standards to provide theanalysis results.